title: HBV DNA replication mediated by cloned patient HBV reverse transcriptase genes from HBV genotypes A-H and its use in antiviral phenotyping assays.
abstract: The aim of this study is to establish a phenotyping assay to analyze patient HBV polymerase/reverse transcriptase (RT) sequences for potential drug resistance against RT inhibitors. HBV RT (pol aa 304-715, including the entire RT) from clinical isolates were amplified and ligated into a plasmid vector (pRTAN) expressing a genotype A HBV genome lacking the RT region. HBV DNA replication of the recombinants and their drug susceptibilities were assessed by transient transfection into HepG2 cells and intracellular core DNA was analyzed either by Southern blot or using a 96-well format and quantification by qPCR. Cloning of the HBV RT gene from clinical isolates representing genotypes A-H was successful and led to virus DNA replication. Recombinants expressing patient derived RT genes containing the rtL180M+M204V lamivudine resistance (LAM-R) mutations demonstrated a LAM-R phenotype. Similarly, patient derived RT genes containing the adefovir resistance (ADV-R) mutations rtA181V or rtN236T demonstrated an ADV-R phenotype. Recombinants containing HBV RT from paired patient samples without genotypic changes had similar EC(50) values. In conclusion, a phenotyping assay for HBV RT gene was developed, allowing evaluation of patient-derived HBV RT from genotypes A-H, and confirmed the drug resistance phenotype in samples containing LAM-R or ADV-R mutations.
authors.affiliation: Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404, United States. yzhu@pharmasset.com
authors.forename: Yuao
authors.initials: Y
authors.lastname: Zhu
authors.forename: Maria
authors.initials: M
authors.lastname: Curtis
authors.forename: Katyna
authors.initials: K
authors.lastname: Borroto-Esoda
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D003001:Cloning, Molecular; D024882:Drug Resistance, Viral; D006515:Hepatitis B virus / Q000187:drug effects*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014779:Virus Replication / Q000187:drug effects*
chemical_list: D000998:Antiviral Agents; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase